Differential calmodulin-modulatory and electron transfer properties of neuronal nitric oxide synthase mu compared to the alpha variant.

Neuronal nitric oxide synthase µ (nNOSµ) contains 34 additional residues in an autoregulatory element compared to nNOSα. Cytochrome c and flavin reductions in the absence of calmodulin (CaM) were faster in nNOSµ than nNOSα, while rates in the presence of CaM were smaller. The magnitude of stimulation by CaM is thus notably lower in nNOSµ. No difference in NO production was observed, while electron transfer between the FMN and heme moieties and formation of an inhibitory ferrous-nitrosyl complex were slower in nNOSµ. Thus, the insert affects electron transfer rates, modulation of electron flow by CaM, and heme-nitrosyl complex formation.

Neuronal nitric oxide synthase is phosphorylated in response to insulin stimulation in skeletal muscle.

Type 2 Diabetes (T2DM) is the seventh leading cause of death in the United States, and is quickly becoming a global pandemic. T2DM results from reduced insulin sensitivity coupled with a relative failure of insulin secretion. Reduced insulin sensitivity has been associated with reduced nitric oxide synthase (NOS) activity and impaired glucose uptake in T2DM skeletal muscle. Upon insulin stimulation, NO synthesis increases in normal adult skeletal muscle, whereas no such increase is observed in T2DM adults. Endothelial NOS is activated by phosphorylation in the C-terminal tail in response to insulin. Neuronal NOS (nNOS), the primary NOS isoform in skeletal muscle, contains a homologous phosphorylation site, raising the possibility that nNOS, too, may undergo an activating phosphorylation event upon insulin treatment. Yet it remains unknown if or how nNOS is regulated by insulin in skeletal muscle. Data shown herein indicate that nNOS is phosphorylated in response to insulin in skeletal muscle and that this phosphorylation event occurs rapidly in C2C12 myotubes, resulting in increased NO production. In vivo phosphorylation of nNOS was also observed in response to insulin in mouse skeletal muscle. These results indicate, for the first time, that nNOS is phosphorylated in skeletal muscle in response to insulin and in association with increased NO production.

Intra- and inter-molecular effects of a conserved arginine residue of neuronal and inducible nitric oxide synthases on FMN and calmodulin binding.

Nitric oxide synthases (NOSs) synthesize nitric oxide (NO), a signaling molecule, from l-arginine, utilizing electrons from NADPH. NOSs are flavo-hemo proteins, with two flavin molecules (FAD and FMN) and one heme per monomer, which require the binding of calcium/calmodulin (Ca(2+)/CaM) to produce NO. It is therefore important to understand the molecular factors influencing CaM binding from a structure/function perspective. A crystal structure of the CaM-bound iNOS FMN-binding domain predicted a salt bridge between R536 of human iNOS and E47 of CaM. To characterize the interaction between the homologous Arg of rat nNOS (R753) and murine iNOS (R530) with CaM, the Arg was mutated to Ala and, in iNOS, to Glu. The mutation weakens the interaction between nNOS and CaM, decreasing affinity by ~3-fold. The rate of electron transfer from FMN is greatly attenuated; however, little effect on electron transfer from FAD is observed. The mutated proteins showed reduced FMN binding, from 20% to 60%, suggesting an influence of this residue on FMN incorporation. The weakened FMN binding may be due to conformational changes caused by the arginine mutation. Our data show that this Arg residue plays an important role in CaM binding and influences FMN binding.

Arginase activity in mitochondria--An interfering factor in nitric oxide synthase activity assays.

Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [(14)C]-L-arginine to [(14)C]-L-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent. Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for L-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [(14)C]-L-arginine to [(14)C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

Proteomic characterization of lipid raft proteins in amyotrophic lateral sclerosis mouse spinal cord.

Familial amyotrophic lateral sclerosis (ALS) has been linked to mutations in the copper/zinc superoxide dismutase (SOD1) gene. The mutant SOD1 protein exhibits a toxic gain-of-function that adversely affects the function of neurons. However, the mechanism by which mutant SOD1 initiates ALS is unclear. Lipid rafts are specialized microdomains of the plasma membrane that act as platforms for the organization and interaction of proteins involved in multiple functions, including vesicular trafficking, neurotransmitter signaling, and cytoskeletal rearrangements. In this article, we report a proteomic analysis using a widely used ALS mouse model to identify differences in spinal cord lipid raft proteomes between mice overexpressing wild-type (WT) and G93A mutant SOD1. In total, 413 and 421 proteins were identified in the lipid rafts isolated from WT and G93A mice, respectively. Further quantitative analysis revealed a consortium of proteins with altered levels between the WT and G93A samples. Functional classification of the 67 altered proteins revealed that the three most affected subsets of proteins were involved in: vesicular transport, and neurotransmitter synthesis and release; cytoskeletal organization and linkage to the plasma membrane; and metabolism. Other protein changes were correlated with alterations in: microglia activation and inflammation; astrocyte and oligodendrocyte function; cell signaling; cellular stress response and apoptosis; and neuronal ion channels and neurotransmitter receptor functions. Changes of selected proteins were independently validated by immunoblotting and immunohistochemistry. The significance of the lipid raft protein changes in motor neuron function and degeneration in ALS is discussed, particularly for proteins involved in vesicular trafficking and neurotransmitter signaling, and the dynamics and regulation of the plasma membrane-anchored cytoskeleton.

Naphthoquinones and bioactive compounds from tobacco as modulators of neuronal nitric oxide synthase activity.

Studies were conducted with extracts of several varieties of tobacco in search of neuronal nitric oxide synthase (nNOS) inhibitors which may be of value in the treatment of stroke. Current therapies do not directly exploit modulation of nNOS activity due to poor selectivity of the currently available nNOS inhibitors. The properties of a potentially novel nNOS inhibitor(s) derived from tobacco extracts, and the concentration-dependent, modulatory effects of the tobacco-derived naphthoquinone compound, 2,3,6-trimethyl-1,4-naphthoquinone (TMN), on nNOS activity were investigated, using 2-methyl-1,4-naphthoquinone (menadione) as a control. Up to 31 microM, both TMN and menadione stimulated nNOS-catalysed L-citrulline production. However, at higher concentrations of TMN (62.5-500 microM), the stimulation was lost in a concentration-dependent manner. With TMN, the loss of stimulation did not decrease beyond the control activity. With menadione (62.5-500 microM), the loss of stimulation surpassed that of the control (78+/-0.01% of control activity), indicating a true inhibition of nNOS activity. This study suggests that potential nNOS inhibitors are present in tobacco, most of which remain to be identified.

Absence of nitric-oxide synthase in sequentially purified rat liver mitochondria.

Data, both for and against the presence of a mitochondrial nitric-oxide synthase (NOS) isoform, is in the refereed literature. However, irrefutable evidence has not been forthcoming. In light of this controversy, we designed studies to investigate the existence of the putative mitochondrial NOS. Using repeated differential centrifugation followed by Percoll gradient fractionation, ultrapure, never frozen rat liver mitochondria and submitochondrial particles were obtained. Following trypsin digestion and desalting, the mitochondrial samples were analyzed by nano-HPLC-coupled linear ion trap-mass spectrometry. Linear ion trap-mass spectrometry analyses of rat liver mitochondria as well as submitochondrial particles were negative for any peptide from any NOS isoform. However, recombinant neuronal NOS-derived peptides from spiked mitochondrial samples were easily detected, down to 50 fmol on column. The protein calmodulin (CaM), absolutely required for NOS activity, was absent, whereas peptides from CaM-spiked samples were detected. Also, l-[(14)C]arginine to l-[(14)C]citrulline conversion assays were negative for NOS activity. Finally, Western blot analyses of rat liver mitochondria, using NOS (neuronal or endothelial) and CaM antibodies, were negative for any NOS isoform or CaM. In conclusion, and in light of our present limits of detection, data from carefully conducted, properly controlled experiments for NOS detection, utilizing three independent yet complementary methodologies, independently as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria.